Bacterial Diversity Profiling by 16S rRNA Metabarcoding

The BCC was determined by metabarcoding of the V3/V4 region of the 16S rRNA gene. Filters dedicated to bacterial diversity were immediately flash-frozen in liquid nitrogen and stored at −80°C until processing. Blank dry filters were sampled simultaneously and used as a contamination control. The two experiments were processed simultaneously. Filters were cut into pieces and DNA was extracted using the NucleoSpin Plant II Mini Kit (Macherey Nagel Ref. 740770.50) according to the manufacturer’s instructions, with an additional lysis step performed for 2 h at 56°C with 25 μL of proteinase K (20 mg mL^–1, Macherey Nagel Ref. 740506) and 100 μL of lysozyme (20 mg mL^–1, Sigma ref 4403-5g). Libraries were prepared and sequenced by Génome Québec using the 341F/785R primers (Klindworth et al., 2013 (link)). Samples were sequenced on an Illumina MySeq using 2 × 300 pb and V3 chemistry. Data were processed using the SAMBA pipeline (v3.0.1)² developed by the IFREMER bioinformatics team (SeBiMER). This resulted in 2,120 amplicon sequence variants (ASVs), which were then clustered using dbOTU3 (Olesen et al., 2017 (link)), resulting in 1,502 ASVs (29% clustering) that were assigned against the Silva v138 database (Quast et al., 2013 (link)).